Patent application title: MONOCYTE-DERIVED STEM CELLS
Glenn E. Winnier (The Woodlands, TX, US)
Brian S. Newsom (Spring, TX, US)
Donna R. Rill (The Woodlands, TX, US)
Jim C. Williams (The Woodlands, TX, US)
Class name: Primate cell, per se human blood, lymphatic, or bone marrow origin or derivative
Publication date: 2010-02-25
Patent application number: 20100047908
Patent application title: MONOCYTE-DERIVED STEM CELLS
Brian S. Newsom
Glenn E. Winnier
Donna R. Rill
Jim C. Williams
POLSINELLI SHUGHART PC
Origin: KANSAS CITY, MO US
Patent application number: 20100047908
Methods for generating multipotent stem cells from adult peripheral blood
monocytes are provided. Monocytes may be de-differentiated into
monocyte-derived stem cells (MDSCs) by contacting the monocyte with the
de-differentation factors, leukocyte inhibitory factor, macrophage
colony-stimulating factor, or a combination thereof. The MDSCs may be
differentiated into many different types of cells upon contact with the
appropriate differentiation factors. Also provided are compositions
comprising the MDSCs or differentiated cells derived from the MDSCs.
1. A method for generating a stem cell, the method comprising:(a)
providing an isolated monocyte; and(b) contacting the monocyte with a
2. The method of claim 1, wherein the de-differentiation agent comprises leukocyte inhibitory factor (LIF) or macrophage colony-stimulating factor (M-CSF).
3. The method of claim 1, wherein the stem cell expresses a marker selected from the group consisting of CD117, DPPA5, HES-1, Oct-4, and SSEA4.
4. The method of claim 1, wherein the monocyte is isolated from mammalian peripheral blood.
5. The method of claim 4, wherein the mammal is a human.
6. The method of claim 5, wherein the human is an adult.
7. The method of claim 1, wherein the monocyte does not express a marker selected from the group consisting of CD117, DPPA5, Oct-4, SSEA-4, CD135, and combinations thereof.
8. The method of claim 1, wherein the stem cell expresses a marker selected from the group consisting of CD117, DPPA5, HES-1, Oct-4, SSEA-4, and combinations thereof.
9. The method of claim 1, wherein the stem cell has a characteristic selected from the group consisting of CD11b+, CD 14+, CD34-, CD45+, CD90-, CD117+, DPPA5+, HES-1+, Oct-4+, SSEA-4+, CD135-, and combinations thereof.
10. The method of claim 1, wherein the monocyte and the stem cell are grown in a serum-free medium.
11. The method of claim 1, wherein the stem cell is generated after 4-8 days in culture.
12. The method of claim 1, wherein the stem cell is contacted with a cryopreservative agent and deep-frozen.
13. An isolated stem cell, wherein the cell expresses a marker selected from the group consisting of CD117, DPPA5, HES-1, Oct-4, SSEA-4, and combinations thereof.
14. The stem cell of claim 13, wherein the stem cell has a characteristic selected from the group consisting of CD11b+, CD14+, CD34-, CD45+, CD90-, CD117+, DPPA5+, HES-1+, Oct-4+, SSEA-4+, CD135-, and combinations thereof.
15. The stem cell of claim 13, wherein the stem cell is derived from an isolated monocyte.
16. The stem cell of claim 15, wherein the monocyte is derived from mammalian peripheral blood.
17. The stem cell of claim 16, wherein the mammal is a human.
18. The stem cell of claim 17, wherein the human is an adult.
19. The stem cell of claim 13, wherein the stem cell is contacted with a cryopreservative agent and deep-frozen.
20. A composition comprising more than 1.times.10.sup.6 of the stem cell of claim 13.
FIELD OF THE INVENTION
This invention relates to methods of generating adult stem cells and compositions of the resultant stem cells.
BACKGROUND OF THE INVENTION
Pluripotent or multipotent stem cells are a valuable resource for research, drug discovery and therapeutic treatments, including transplantation (Lovell-Badge, 2001, Nature, 414:88-91; Donovan et al., 2001, Nature, 414:92-97; Griffith et al., 2002, Science, 295:1009-1014; Weissman, 2002, N. Engl. J. Med., 346:1576-1579). These cells, or their mature progeny, can be used to study signaling events that regulate differentiation processes, identify and test drugs for lineage-specific beneficial or cytotoxic effects, or replace tissues damaged by disease or an environmental impact. The current state of stem cell biology and the medicinal outlook, however, are not without drawbacks or free from controversy.
The use of pluripotent or multipotent stem cells from fetuses, umbilical cords or embryonic tissues derived from in vitro fertilized eggs raises ethical and legal questions in the case of human materials, poses a risk of transmitting infections and/or may be ineffective because of immune rejection. In particular, embryonic stem cells have a number of disadvantages. For example, embryonic stem cells may pass through several intermediate stages before becoming the cell type needed to treat a particular disease. In addition, embryonic stem cells may be rejected by the recipient's immune system since it is possible that the immune profile of the specialized cells would differ from that of the recipient.
One way to circumvent these problems is by exploiting autologous stem cells, preferably from an easily accessible tissue such as peripheral blood. The most widely used source of adult stem cells is derived from bone marrow or peripheral blood. The mesenchymal compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into a variety of different cell types including adipogenic, osteogenic, chondrogenic, and myogenic cells when cultured under the appropriate growth conditions (Pittenger et al., 1999, Science, 284:143-147). Early studies using bone marrow stromal cells for tissue repair focused on the repair of bone defects (Takagi and Urist, 1982, Clin Orthop, 171:224-231). However, more recent studies have applied bone marrow stem cells to repair a variety of damaged tissue types, including cartilage (Wakitani et al., 2002, Osteoarthritis Cartilage, 10: 199-206), myocardium (Orlic et al., 2003, Pediatr Transplant, 7 Suppl 3:86-88; Terai et al., 2002, J Gastroenterol, 37 Suppl 14:162-163), and most recently diabetes (lanus et al., 2003, J Clin Invest, 2003; 111:843-850). Recent studies have demonstrated that bone marrow contains cells that appear to have the ability to trans-differentiate into mature cells belonging to cell lineages other than those of the blood (Laggase et al., 2000, Nature Med, 6:1229-1234; Orlic et al., 2001, Nature, 410:640-641; Korbling et al., N. Engl J Med, 346:738-746). However, recent studies have suggested that these cells undergo a trans-differentiation that results from the fusion of the stem cell with resident tissue cells (Terada et al., 2002, Nature, 416:542-545; Ying et al., 2002, Nature, 416:545-548). But, autologous bone marrow procurement has potential limitations including low yields, costly processes, and painful procedures. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous.
Thus, needs exist in the art to isolate, culture, sustain, propagate, and differentiate adult stem cells, particularly human adult stem cells that are relatively accessible in order to develop cell types suitable for a variety of uses. Such uses may include the use of autologous stem cells for the treatment of diseases and amelioration of symptoms of diseases.
SUMMARY OF THE INVENTION
Provided herein is a method for generating a stem cell. A monocyte may be contacted with a de-differentiation factor, such as leukocyte inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), or a combination thereof, which may cause the monocyte to de-differentiated into a stem cell. The monocyte may be isolated from mammalian peripheral blood. The mammal may be a human. The mammal may be an adult.
The stem cell may be generated after 4-8 days in culture. A plurality of stem cells may be generated. The plurality of cells may comprise more than 1×106 cells. The stem cell may be contacted with a cryopreservative agent and deep-frozen.
The stem cell may express CD117, DPPA5, HES-1, OCT-4, SSEA4, or a combination thereof. The monocyte may not express CD117, DPPA5, Oct-4, SSEA-4, or a combination thereof. The stem cell may have any of the following characteristics: CD4+, CD11b+, CD14+, CD45+, CD90-, CD117+, DPPA5+, HES-1+, Oct-4+, SSEA-4+, CD34-, CD135- or a combination thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts graphs illustrating the growth of monocyte-derived stem cells in different medium formulations and different concentration of fetal bovine calf serum (FBS). Panel A presents the percent confluency on day 3 and panel B presents the percent confluency on day 6.
FIG. 2 depicts a graph illustrating the total cell count in three different preparations of monocyte-derived stem cells from days 1 to 15 of culture.
FIG. 3 depicts a graph illustrating the average cell diameter in two different preparations of monocyte-derived stem cells from days 1 to 8 of culture.
FIG. 4 depicts DNA histograms of monocyte-derived stem cells. Panel A presents the DNA profile of large adherent cells (MDSCs) on day 2 of culture, and panel B present the DNA profile of large adherent cells (MDSCs) on day 6 in culture. The percent of cells in each phase of the cell cycle is presented below each histogram.
FIG. 5 depicts graphs illustrating the percentage of cells in each phase of the cell cycle from days 2 to 6. Plotted are the percentage of small non-adherent cells (NA), large NA, small adherent (Ad), and large Ad cells. Panel A presents the percentage of cells in G0/G1 phase. Panel B presents the percentage of cells in S phase. Panel C present the percentage of cells in G2/M phase. Panel D presents the percentage of aneuploid cells.
FIG. 6 depicts photomicrographs of the expression of cell lineage markers in monocyte-derived stem cells on day 6. Cell nuclei were stained with DAPI (blue). Panel A shows low expression of CD14 (green). Panel B shows no expression of CD34. Panel C shows no expression of CD90. Panel D shows no expression of Nestin. Panel E shows high expression of HLA. Panel F shows low expression of osteocalcin.
FIG. 7 depicts photomicrographs of the expression of stem cell markers in monocyte-derived stem cells on day 5. Cell nuclei were stained with DAPI (blue). Panel A shows expression of HES 1 (green). Panel B shows expression of SSEA4. Panel C shows expression of CD117. Panel D shows control cells.
FIG. 8 depicts a graph illustrating the relative expression of specific genes in monocyte-derived stem cells from day 1 to day 15. Gene expression was analyzed by real-time PCR.
FIG. 9 depicts a histograms showing the expression of CD11b (A), CD 135 (B), CD14 (C), and CD 123 (D) (dotted black lines), compared to IgG (solid dark line, A-D) in buffy coat #49 of MDSCs at day 9 compared to IgG as measured using antibody staining and flow cytometry.
FIG. 10 depicts a histograms showing the expression of CD11b (A), CD135 (B), CD14 (C), and CD123 (D) (dotted black lines), compared to IgG (solid dark line, A-D) in buffy coat #66 of MDSCs at day 21 cultured in de-differentiation medium as measured using antibody staining and flow cytometry.
DETAILED DESCRIPTION OF THE INVENTION
1. A Method for Generating a Stem Cell
Provided herein is a method for generating a multipotent stem cell. The stem cell may be generated by contacting a monocyte with a de-differentiation factor. The de-differentiation factor may be leukocyte inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), and a combination thereof. Exposure to the de-differentiation factor may cause the monocyte to de-differentiate into a stem cell. The stem cell may express a marker, such as CD117, DPPA5, HES-1, Oct-4, SSEA-4, and combinations thereof.
The monocyte may be derived from peripheral blood, which may be from a mammal. The mammal may be a human, a research animal, or a domesticated livestock or pet. The mammal may be an adult.
The monocyte may be derived from peripheral blood using, for example, a single-step discontinuous Ficoll gradient fractionation procedure. The monocyte may be isolated from peripheral blood using another method known to a skilled artisan. The monocyte may be freshly isolated or may be from a frozen preparation.
b. Growth and De-differentiation
The monocyte may be grown in a culture medium. The culture medium may be AIM V (Invitrogen). The monocyte may be seeded on coated or uncoated polystyrene culture plates, dishes, or slides. The culture vessel may be coated with fibronectin, gelatin, collagen, polylysine, or L-ornithine. The cells may be seeded on untreated FALCON integrid vacuum-gas plasma treated plates or dishes. The density of cells to be seeded may range from approximately 1×106/ml to approximately 2×106/ml.
The monocyte may be contacted with leukocyte inhibitory factor (LIF) and macrophage colony-stimulating factor (M-CSF). The concentration of LIF may be from approximately 10 ng/ml to approximately 25 ng/ml. The concentration of M-CSF may range from approximately 5 ng/ml to approximately 50 ng/ml. For example, the concentrations of LIF and M-CSF may be 10 ng/ml and 25 ng/ml, respectively. The de-differentiation factors, LIF and M-CSF, may be provided to the cells in the presence of a culture medium.
The culture medium may be LDMEM (low glucose DMEM), HDMEM (high glucose DMEM), DMEM/F12, or Megacell DMEM/F12. The culture medium may be supplemented with 10-20% fetal bovine calf serum (FBS). Cultures may also be supplemented with 10-20% human AB serum.
The cultures may also be grown in serum free conditions. The growth and de-differentiation of cells may be conducted using Megacell DMEM/F12 medium without FBS (fetal bovine serum). The medium may be supplemented with sodium selenite, rh-Insulin, human transferrin, fatty acids, 4,500 mg/L D-glucose, 4 mM L-glutamine, penicillin-streptomyocin, and combinations thereof. Other media in similar serum-free conditions may be utilized
M-CSF and LIF may be natural or synthetic, and may be used in a purified or unpurified state. Further, the M-CSF or LIF may be a holoprotein or may be active subunits or fragments that exhibit a mitogenic effect on isolated monocytes. Conventional titration assays may be used to determine the effective concentration of M-CSF or LIF.
The monocyte may be cultured under growth conditions well known in the art to propagate the cells, such as 37° C., 5% CO2. The culture medium containing LIF and M-CSF may be changed every three days. Cell growth and de-differentiation parameters may be analyzed by dispersing and collecting the cells. The cells may be dispersed by addition of approximately 0.5% lidocaine with gentle scraping. The cells may also be dispersed by addition of trypsin/EDTA or collagenase with gentle scraping. The dispersed cells may be counted using a cell counter, examined under a microscope, stained for cell markers, or used for molecular analyses.
c. Monitoring De-Differentiation
The de-differentiation of a monocyte into a stem cell may be monitored by a variety of methods well known in the art. Changes in a parameter between an untreated control cell and a LIF/M-CSF-treated cell may be an indication that the cell has de-differentiated. Changes in the rate of proliferation may indicate de-differentiation. A control monocyte may be essentially quiescent, whereas a de-differentiated cell may have an increase in the rate of cell proliferation. Changes in the rate of proliferation may be measured by counting the total number of cells in the two populations. Changes in the cell cycle may also indicate that the cells have undergone a de-differentiation process. A control monocyte may be in the GO/GI phase of the cell cycle, whereas a cell undergoing de-differentiation may be in the S or G2/M phases of the cell cycle. Changes in the cell cycle may be monitored by flow cytometry. Changes in the cell cycle also may be monitored by the incorporation of BrdU into newly synthesized DNA or by staining for a cell proliferation antigen, such as PCNA or cyclins.
Changes in the expression of a specific marker may also indicate de-differentiation. Expression of specific markers may be monitored at the level of protein by staining with antibodies against the marker. Cell surface or intracellular markers that may be examined include, but are not limited to, CD3, CD11b (MAC-1), CD14, CD31, CD34, CD45, CD90 (Thy-1), CD117 (c-kit receptor), CD123 (IL3R), CD133, 135 (Flk-2), DPPA4, HES-1, HLAabc, MAP-2, nestin, Oct-4, osteocalcin, pankeratin, SSEA4, VEGF-R3, VEGFR (KDR), and vWF. The cells may be fixed and immunostained using procedures well known in the art. For example, a primary antibody may be labeled with a fluorophore or chromophore for direct detection, or a primary antibody may be detected with a secondary antibody that is labeled with a fluorophore or chromophore. The fluorophore may be fluorescein, FITC, rhodamine, Texas Red, Cy-3, Cy-5, Cy-5.5, Alexa488, Alexa594, QuantumDot525, QuantumDot565, or QuantumDot655. The fluorescently labeled cell may be examined under a fluorescent light microscope, a confocal microscope, or a multi-photon microscope. The labeled cell may also be analyzed by flow cytometry
RT-PCR and quantitative PCR methods may be used to monitor the changes in gene expression. RNA may be isolated from the cells using procedures known to one skilled in the art. Similarly, PCR may be performed using conditions and parameters well known in the art. Gene transcripts that may be amplified during PCR include ABCG2, AC133, ACTB, AFP, ALB, ANF, ATP2A2, BMP-4, BNP, carboxypeptidase, CD4, CD9, CD10, CD11B, CD13, CD14, CD31, CD33, CD34, CD38, CD45, CD90 (THY1), CD105, CD117 (c-kit receptor), CD123 (IL3R), CD133, CD135 (Flk-2), CDX-2, CK18, CK19, col2a1, CXCR3, CXCR4, DPPA5, E-cadherin, Flk-1, GAD, GAPDH, GATA-2, GATA-3, GATA4, GENESIS, GFAP, GLP-1R, glucagon, Glut2, HLA-A, HNF-3B, IAPP, IGF2, insulin, IPF1, GLP-1, Islet1, keratin, MAP2, MBP, myosin heavy chain, nestin, neurogenin, NGN3, NKX-2.2, NKX2.5, NSE, Oct4, osteocalcin, osteopontin, pancreatic amylase, PAX-4, PAX6, PDGFRB, PDX-1, PPAR2, REX-1, SCF, SM1, SM22A, somatostatin, SOX-2, TAL-1, TAU, TBX-5, TIE-2, troponin, VE-cadherin, and VEGFR2 (KDR). Changes in gene expression (increases or decreases) between two cells exposed to different conditions may indicate that the state of differentiation has changed between the two cells.
The expression of some cell markers may not change during differentiation. Markers whose expression may be detected in both monocytes and stem cells include, AC133, ANF, BMP-4, BNP, CD4, CD9, CD10, CD11b, CD14, CD31, CD33, CD45, CD71, CD90, CD123 (IL3R), CD133, CD135 (Flk-2), CK18, CK19, C-peptide, CXCR3, GATA4, GLUT2, HLAabc, IAPP, Islet-1, osteopontin, and PDX-1. However, the expression of some of these markers may increase or decrease during the cells differentiation. Markers whose expression may not be detected in both monocytes and stem cells include CD3, CD8, CD 19, CD20, CD34, CD80, CD86, glycophorin A, MAP2, nestin, pankeratin, and vWF. The expression of certain markers may increase in a stem cell relative to a monocyte. Stem cell-specific markers that may increase include CD117, DPPA5, HES-1, Oct-4, SCF, and SSEA-4. The stem cell may be CD34-.
d. Identifying a De-Differentiated Cell
The de-differentiation of the monocyte into a multipotent stem cell may be identified by alternations in the rate of cell proliferation, cell cycle, or gene expression when cultured under specific conditions. After approximately 4-8 days in culture, the confluency of the LIF/M-CSF-treated cell may be greater than 75%, 80%, 85%, 90%, or 95%. After approximately 3 days in culture, the monocyte may have de-differentiated into a stem cell, as evidenced by changes in cell proliferation and gene expression. The percentage of de-differentiated cells in a population of cells may be at least 40, 50, 60, 70, 80, or 90% of the total number of cells. The population of stem cells may be maintained by continued culture in the presence of the growth factors, LIF and M-CSF.
The stem cell may be preserved indefinitely by contacting the cell with a cryopreservative agent and freezing the cell. The frozen cell may be stored at an ultra low temperature or in liquid nitrogen.
2. Using the Stem Cell
The stem cell may be differentiated into another cell type by the addition of the appropriate growth factors or hormones. As an example, the stem cell may be differentiated into a neuronal cell by contact with NGF, brain-derived neurotrophic factor, neurotrophin-3, basic fibroblast growth factor, pigment epithelium-derived factor, retinoic acid, and combinations thereof. The stem cell may be differentiated into an endothelial cell by contact with VEGF, IGF, BFGF, and combinations thereof. The stem cell may be differentiated into an epithelial cell by contact with EGF, BMP-4, activin, elevated calcium concentrations, retinoic acid, sodium butyrate, vitamin C, hexamethylene bis acetate, phorbol 12-myristate 13-acetate (PMA), teleocidin, interferon gamma, staurosporin, and combinations thereof. The stem cell may be differentiated into a macrophage or a T cell by contact with LPS, IL-2, IL-4, IL-12, IL-18, CD3 antibody, PMA, teleocidin, interferon gamma, and combinations thereof. The stem cell may be differentiated into a hepatocyte by contact with HGF, retinoic acid, oncostatin M, phenobarbital, dimethyl sulfoxide, dexamethasone, dibutyryl cyclic AMP, and combinations thereof.
The expression of cell lineage-specific markers may increase in a stem cell relative to a monocyte. Non-limiting examples of embryonal carcinoma (EC)-specific markers that may increase over time include Flk-1, TIE-2, and VE-cadherin. Non-limiting examples of hematopoietic-specific markers that may increase over time include TAL-1, GATA-2, and GATA-3. Non-limiting examples of cardiac-specific markers that may increase over time include NKX2.5, NKX2.2, and CD105. Non-limiting examples of pancreatic-specific markers that may increase over time include IPF1, insulin, PAX-4, IGF2, and glucagon. Non-limiting examples of endoderm-specific markers that may increase over time include AFP and ALB. Non-limiting examples of smooth muscle-specific markers may typically increase over time include SM1, SM22A, and PDGFRB.
The other cell types or differentiated cells derived from the stem cell may be used, by way of non-limiting example, to replenish or stimulate (induce) the replenishment of a cell population that has been reduced or eradicated by a disease or disorder (e.g., cancer), to treat a disease or disorder (e.g., a cancer therapy), or to replace damaged or missing cells or tissue(s). By way of example, neuronal tissue damaged during the progression of Parkinson's disease, endothelial cells damaged by surgical incisions, macrophage cells affected by Gaucher's disease, epithelial cells damaged from skin burns, hepatocytes damaged as a result of cirrhosis, pancreatic islet β-cells damaged by type I diabetes, or cardiac cells damaged by heart disease may be replenished or stimulated to replenish cells differentiated from these stem cells. Moreover, since these stem cells may be derived from the peripheral blood of the same individual who will later receive the stem cell or their derivatives, immunosuppression may not be necessary.
Also provided herein are compositions comprising the stem cell or differentiated cells derived from the stem cell. The composition may comprise a plurality of the stem cell, which may be more than 1×106 of the stem cell. The stem cell may express a marker, which may be CD117, DPPA5, HES-1, Oct-4, SSEA-4, or a combination thereof. The stem cell may have a characteristic, which may be CD11b+, CD14+, CD34-, CD45+, CD90-, CD117+, DDPA5+, HES-1+, Oct-4+, SSEA-4+, CD135-, or a combination thereof.
As various changes could be made in the above compounds, methods, and products without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.
Generation of Monocyte-Derived Stem Cells (MDSCs)
Isolation of Monocytes. Monocytes were isolated from adult human peripheral blood using a single-step discontinuous Ficoll gradient. During this procedure, peripheral blood monocytes are localized to the interface between the blood plasma and the separation medium. To help maintain the interface, LeucoSep centrifuge tubes, which contain a positioned porous membrane barrier, were used. LeucoSep tubes (30-ml) were prepared by adding 15 ml of Lymphocyte Separation Buffer (Cat. no. 25-072-cv, Mediatech Cellgro) and centrifuging at 1000×g for 30 sec at room temperature to drive the buffer through the membrane barrier. Then 15 ml of blood and 30 ml of 1×HBSS (Hanks Balanced Salt Solution) with 2 mM EDTA were added to each tube. The tubes were centrifuged at 1000×g for 10 minutes at 4° C. After this centrifugation step, the enriched cell fraction containing lymphocytes and monocytes was located above the membrane barrier. The tubes were carefully removed from the rotor to minimize disruption of the layers. The enriched cell fraction was carefully removed with a Pasteur pipette and transferred to a 50-ml centrifugation tube and the tube filled to 50 ml with 1×HBSS that does not include Ca2+ and Mg2+ (Cat. No. 21-022-cm, Mediatech Cellgro).
The cells were centrifuged at 150×g for 15 minutes at room temperature, and the supernatant was removed. Then 10-15 ml of Red Blood Cell Lysis Buffer (Cat. No. R7757, Sigma-Aldrich) was added to the pelleted cells to remove any red blood cells that may contaminate the mononuclear cell layer. After 2 minutes, 40 ml of 1×HBSS was added to the cells, which were then spun at 150×g for 15 minutes at room temperature. The cell pellet was washed two more times with 50 ml of 1×HBSS to remove residual lysis buffer. The final pellet was resuspended in AIM V medium (Invitrogen), which is a serum-free medium that contains L-glutamine and streptomycin sulfate at 50 μg/ml. Cell density was determined using a Vi-CELL Cell Analyzer (Beckman Coulter).
De-differentiation into Monocyte-Derived Stem Cells (MDSCs). The isolated monocytes were seeded on a variety of plate formats at a density of 1-2×106/ml. At this density, the cells were >75% confluent after 6 days in culture. Table 1 presents the different plates and total number of cells when plated at a density of 1×106 cells/cm2. The cells were plated in a 2:1 mixture of Megacell DMEM/F12 medium (Cat. No. M4192, Sigma-Aldrich) and AIM V medium and cultured overnight at 37° C. and 5% CO2. The Megacell DMEM/F12 medium is a serum-free media based on the standard published basal formulation, but is further supplemented with buffers and sodium pyruvate. Sodium selenite, rh-Insulin, human transferrin, and fatty acids have been added to allow for serum reduction. It contains 4,500 mg/L D-glucose. Generally, it was further supplemented with 4 mM L-glutamine and penicillin-streptomyocin prior to use.
After 24 hours, the culture medium was removed and the cells were gently washed three times with 1×HBSS containing 2 mM EDTA. De-differentiation medium was added. The de-differentiation medium consisted of Megacell DMEM/F12 or LDMEM (low glucose DMEM) or HDMEM (high glucose DMEM) containing 10 ng/ml leukocyte inhibitory factor (LIF; Cat. No. LIF1010, Chemicon) and 25 ng/ml macrophage colony-stimulating factor (M-CSF; Cat. No. GF053, Chemicon). After three days, the medium was removed and replaced with fresh de-differentiation medium. After 6 days in culture the cells had de-differentiated into monocyte-derived stem cells. Cultures grown for longer than 10 days tended to develop into multinucleated osteoclastic giant cells and endothelial cells. Cells grown in the absence of LIF and M-CSF remained quiescent and did not de-differentiate.
TABLE-US-00001 TABLE 1 Total cells plated on the different types of plates. Dish type Area Cell Density Total Cells 15 cm plate 176 cm2 1 × 106 cells/cm2 176 × 106 cells/plate 10 cm plate 78 cm2 1 × 106 cells/cm2 78 × 106 cells/plate 6 well dish 9.5 cm2 1 × 106 cells/cm2 9.5 × 106 cells/well 24 well dish 1.9 cm2 1 × 106 cells/cm2 2.0 × 106 cells/well 48 well dish 1.1 cm2 1 × 106 cells/cm2 1.1 × 106 cells/well 1 well chamber 8.6 cm2 1 × 106 cells/cm2 8.6 × 106 cells/well slide 4 well chamber 1.7 cm2 1 × 106 cells/cm2 1.7 × 106 cells/well slide 8 well chamber 0.7 cm2 1 × 106 cells/cm2 0.7 × 106 cells/well slide
Dispersion, Freezing and Thawing of MDSCs. Adherent cells (known as MDSCs) were removed by treating the cultures with 0.5% lidocaine for 1-2 minutes. Concentrations of lidocaine greater than 1% caused an increase in cell death and a decrease in the overall cell proliferation rate. (Trypsin/EDTA and collagenase were also used to disperse the cells.) The cells were dispersed by gentle scraping and transferred to a new tube. Two volumes of Megacell DMEM/F12 or LDMEM or HDMEM were added to neutralize the lidocaine and the cells were centrifuged at 150×g for 15 minutes at room temperature. The supernatant was removed and fresh Megacell DMEM/F12 or LDMEM or HDMEM was added. To freeze the cells, 500 μl of DMSO Freezing Medium (Cat. No. 210002, Bioveris Corp.) was added to a 500 μl aliquot of 1×106 cells. The tube was mixed well, frozen in an ethanol-freezing chamber, and placed at -80° C. overnight. The tube was transferred to liquid nitrogen for long-term storage. To thaw the cells, a vial of frozen cells was gently swirled in a 37° C. water bath and the cells were transferred to a 15-ml tube. Four ml of Megacell DMEM/F12 or LDMEM or HDMEM at room temperature (approximately 22° C.) was slowly added and gently mixed by swirling. The cells were spun at 150×g, the supernatant was removed, and the cells were resuspended in 2.5 ml of culture medium. The cells were ready to be plated and cultured. Cell viability was typically >90%.
Growth in Different Medium Formulations
To determine the optimal conditions for growth and de-differentiation, several different medium compositions and serum levels were examined. Monocytes were derived as described in Example 1; they were plated in AIM V medium and cultured overnight at 37° C. The cells were then transferred to and grown in five different medium formulations: HDMEM, LDMEM, AIM V, RPMI, or IN VIVO 15 media. Each formulation was supplemented with 0, 5, 10, or 20% FBS and the two de-differentiation agents, 10 ng/ml LIF and 25 ng/ml M-CSF. The cells were grown for 6 days, with the medium changed at day 3. There was no difference in the percentage of MDSCs among the different conditions, but the total number of cells varied significantly among the different conditions. As shown in FIGS. 3A and 3B, growth in the presence of LDMEM or HDMEM and 10-20% FBS resulted in much higher total number of total cells per plate.
Growth on Different Substrates
To determine whether the substrate affected growth and de-differention, isolated monocytes were plated on fibronectin, gelatin, collagen, poly-lysine, or L-ornithine coated plates. The cells were grown in de-differentiation medium for 6 days, with the medium changed at day 3. Cells were collected by treatment with 0.5% lidocaine with gentle scraping and counted with a Vi-CELL cell counter. There was a small increase in the total numbers of cells grown on fibronectin or gelatin-coated plates (5-15% increase in total cell number) after 3 and 6 days in culture. The percentage of MDSCs was not significantly changed among the different treatments.
When culturing cells on different brands of polystyrene tissue dishes, it was discovered that there was a 50% increase in the initial adhesion and growth of cells on FALCON integrid vacuum gas plasma treated plates, as compared to NUNC and other brands of plates. There was also a higher percentage of MDSCs generated on the FALCON plates, e.g., 90% on FALCON plates compared to approximately 50% on NUNC plates at the same time point.
Cell Growth and Cell Size Analysis
To characterize the growth and proliferation of MDSCs, the total cell numbers and average cell diameters were determined. Several different preparations of monocytes were isolated essentially as described in Example 1 and grown in the presence of de-differentiation medium for 12-15 days, with the medium changed every three days. There was an increase in total number of cells during the de-differentiation phase (day 1 to day 6), after which the cell count decreased (FIG. 2). The diameter of the cells increased from approximately 9-10 microns to approximately 16 microns during the first 8 days in culture, after which the size of the cells stabilized (FIG. 3).
Cell Cycle Analysis
To examine changes in the cell cycle as the monocytes de-differentiated into MDSCs, flow cytometry was used. This analysis also provided the opportunity to examine the growth and de-differentiation of MDSCs during long term culturing. For these experiments, monocytes were grown in 6-well plates in the presence of de-differentiation medium for 6 days, and cells were removed from individual wells at various time points for analysis. The following cell types were characterized: small non adherent, large non adherent, small adherent, and large adherent. Panels A and B of FIG. 4 present the cell cycle analysis of large adherent cells (also known as MDSCs) on day 2 and day 6, respectively. At day 2, the cells were quiescent, with >99% in the G1/G0 phase. By day 6, a significant percentage of cells had re-entered the cell cycle, as evidenced by the increased percentages of cells in S or G2/M phases of the cell cycle. Binucleated cells were identified mainly in the G2/M phase of the cell cycle; these cells were composed of greater than 1 nuclei per cell. However, cells that contained greater than 4n of nuclei DNA were classified as aneuploid.
FIG. 5 shows a detailed analysis of the percent of each type of cell in the different phases of the cell cycle during days 2-6 of the de-differentiation process. By days 5-6 there is a shift in the percentage of cells in S and G2/M phases of the cycle. The percentage of aneuploid cells also increased over time. The growth analysis (see Example 4) and this cell cycle analysis suggest that the MDSCs generated by this procedure were consistent with the characteristics of a population of slowly dividing cells.
Phenotypic Analysis: Flow Cytometry
To characterize the phenotypic profiles of the MDSCs during their growth and de-differentiation, they were stained for cell lineage-specific and stem cell-specific markers. Monocytes were collected and cultured (up to 25 days) essentially as described in Example 1. At each time point, cells were collected, washed, and resuspended in Staining Buffer (1×PBS with 1% FBS and 0.1% sodium azide) at a concentration of 1×107 cells/ml. Up to 1×106 cells were used per staining reaction in a final volume of 100-200 μl. Some cells were only stained for extracellular antigens. For these, the antibodies were diluted in Staining Buffer at the appropriate concentration (see Table 2) and added to the above-prepared cells. The tube was gently mixed and incubated for 15 minutes at room temperature in the dark. The cells were washed in 2 ml of ice-cold Staining Buffer and centrifuged for 6 minutes at 300×g. If this was the only antibody used, the cell pellet was resuspended in 200 μl of 2% paraformaldehyde and stored at 4° C.
Other cells were stained for intracellular antigens only or a mixture of extracellular and intracellular antigens. For these, the cells were fixed and permeabilized by resuspending the washed cell pellet in 2 ml of FACSLyse (Becton Dickinson). The cells were incubated for 10 minutes at room temperature in the dark, and then washed with 2 ml of ice-cold Staining Buffer. After centrifugation at 300×g for 6 minutes, the supernatant was discarded and the pellet was resuspended in 0.5 ml of FACS Permeabilization Buffer II (Becton Dickinson). The cells were incubated for 10 minutes at room temperature in the dark, and then washed with 2 ml of ice-cold Staining Buffer. After centrifugation at 300×g for 6 minutes, the supernatant was discarded and the pellet was resuspended in 100 μl of Staining Buffer. The appropriate antibodies were added at the appropriate concentration (Table 2), mixed well, and incubated for 30 minutes at room temperature in the dark. The cells were washed with 2 ml of ice-cold Staining Buffer, spun at 300×g for 6 minutes, and the cell pellet was resuspended in 300 μl of Staining Buffer. The cells were then analyzed by flow cytometry.
TABLE-US-00002 TABLE 2 Directly conjugated antibodies for flow cytometry Antibody Vendor Catalog Number Dilution ABCG2 APC R&D Systems FAB995A 1:5 GlycoPhorin A PE Becton Dickinson 340946 1:10 CXCR3 PE Pharmingen 557185 1:10 CD3 PerCP Becton Dickinson 340663 1:10 CD4 PE Becton Dickinson 340670 1:10 CD8 FITC Becton Dickinson 340692 1:10 CD10 FITC Becton Dickinson 340924 1:10 CD11b PE Becton Dickinson 340712 1:10 CD14 PerCP Becton Dickinson 340660 1:10 CD14 FITC Becton Dickinson 347493 1:10 CD15 FITC Becton Dickinson 340703 1:10 CD19 PerCP-Cy5.5 Becton Dickinson 340951 1:10 CD20 FITC Becton Dickinson 340673 1:10 CD33 PerCP-Cy5.5 Becton Dickinson 341640 1:10 CD34 PE Becton Dickinson 340669 1:10 CD45 APC Becton Dickinson 340942 1:20 CD71 FITC Becton Dickinson 340717 1:10 CD80 PE Becton Dickinson 340294 1:10 CD86 CyChrome Pharmingen 555666 1:10 CD117 PE Becton Dickinson 340867 1:10 CD133 APC Miltenyi Biotech 120-001-123 1:5
The cells were stained for a variety of stem cell-specific and cell lineage-specific markers. A summary of the expression profile during the de-differentiation phase (day 2-6) is presented in Table 3. A summary of the long-term patterns of expression (days 5-25) of these markers is presented in Table 4. Some monocytic and hematopoietic markers (e.g., CD11b/MAC-1, CD14, CD45) are expressed in these MDSCs from the onset and throughout the culture period. CX34 expression was not detected in either short- or long-term cultures.
TABLE-US-00003 TABLE 3 Short-term phenotypic expression as revealed by flow cytometry. d2NA d2Ad d3 NA d3Ad d4 NA d4 Ad d5 NA D5 Ad d6 NA d6 Ad CD3 - - - - - - - - - - CD4 + + + + + + + + + + CD8 - - - - - - - - - - CD10 - - - - - - - - - - CD11b (MAC-1) +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ CD14 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ CD15 - - - - - - - - - - CD19 - - - - - - - - - - CD33 - - - - - - - - - - CD34 - - - - - - - - - - CD45 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ CD71 (transferrin receptor ++ ++ ++ ++ + + + + + + CD90 (Thy1) - - - - - - - - - - CD117 (c-kit receptor) - - - - - - - - - - CD133 - - - - - - - - - - ABCG2 - - - - - - - - - -
TABLE-US-00004 TABLE 4 Long-term phenotypic expression as revealed by flow cytometry. Marker d5 d10 d15 d19 d25 CD3 - - - - - CD4 + + + + + CD8 - - - - - CD11b +++ +++ +++ +++ +++ CD14 +++ +++ +++ +++ +++ CD20 - - - - - CD33 - - - - - CD34 - - - - - CD45 ++ ++ ++ ++ ++ CD71 (Transferrin ++ ++ ++ ++ + CD80 - - - - - CD86 - - - - - CD90 (Thy1) - - - - - CD117 (c-kit R) - - - - - CD133 - - - - - ABCG2 - - - - - Glycophorin A - - - - -
The phenotypic profile of MDSCs was further characterized during growth and differentiation by examining the expression of several other markers. MDSCs were isolated and cultured in a 6 well dish format in de-differentiation medium containing LIF and M-CSF as described above. MDSCs were then stained with antibodies against CD11b (MAC-1), CD14, CD123 (IL3R), and CD135 (Flk-2), and then analyzed by flow cytometry at day 9 (FIG. 9) and day 21 (FIG. 10).
FIG. 9 shows that MDSCs expressed high levels of CD11b (FIG. 9A) and CD14 (FIG. 9C), consistent with marker expression in a monocyte lineage. MDSCs also expressed CD123 at day 9 (FIG. 9D). MDSCs did not express CD135, suggesting a lack of Flk-2 expression (FIG. 9B).
FIG. 10 shows that, consistent with a monocyte lineage, MDSCs expressed high levels of CD11b (FIG. 10A) and CD14 (FIG. 10C) at day 21. In contrast to day 9, MDSCs expressed low levels of CD 123 at day 21 (FIG. 10D). As at day 9, MDSCs did not express CD 135 at day 21 (FIG. 10B).
In summary, high-levels of CD11b and CD14 were expressed in MDSCs at all time points measured, and CD135 (Flk-2) expression was absent in MDSCs at all time points measured. CD123 (IL3R) was positive early during de-differentiation (day 9), and lost expression intensity over time. By day 21, cultured MDSCs exhibited barely detectable levels of CD123.
Phenotypic Analysis: Immunofluorescence Staining
To better visualize the time course of activation of stem cell-specific markers, in particular, immunofluorescence staining was performed. Monocytes were isolated and cultured in 8-chamber slides using the method described in Example 1. For each time point, cells were collected, washed in Wash buffer (PBS+1% BSA) to remove any remaining medium. The cells were fixed in 200 μl of freshly made 4% formaldehyde (in PBS) for 20 minutes at room temperature, and then washed in Wash Buffer. Cells were permeabilized by adding 200 μl of FACS Permeabilization Buffer II, incubating for the appropriate time at room temperature, and washing three times in Wash Buffer. Cells were stained for specific markers by incubating with primary antibodies diluted in 200 μl of Wash Buffer (see Table 5) for 3-4 hours at room temperature, washing three times in Wash Buffer, incubating with diluted secondary antibodies for 1 hour at room temperature in the dark, and washing three times in wash buffer. Incubating in 100 μl of 10 μg/ml DAPI for 5 minutes at room temperature stained the DNA of the cells, the cells were then washed three times in wash buffer to remove any residual DAPI stain. After washing cells, an anti-fade reagent was then added to the cells to enhance fluorescent detection.
Cells stained only for extracellular markers were fixed, stained with antibodies, permeabilized for 5 min, and stained with DAPI. Cells stained only for intracellular markers or for both intra/extracellular markers were fixed, permeabilized for 30 minutes, stained with antibodies, and stained with DAPI.
Table 6 summarizes the phenotypic expression patterns during the de-differentiation phase. The expression of three stem cell-specific markers (i.e., HES-1, SSEA4, and CD117) increased over time. Initially, these markers had no or low levels of expression, but their levels increased beginning around days 4. HES-1 and SSEA4 are primitive stem cell markers, and CD117 (c-kit receptor) is normally expressed by stem cells and during embryogenesis. Table 7 presents the long-term patterns of expression of these lineage- and stem cell-specific markers. Together, these data and the flow cytometry data revealed that CD14, CD45, and CD11b expression remained constant, and CD34 was never detected in these MDSCs.
FIG. 5 presents images of cells stained for lineage-specific markers at day 9. The cells had low levels of CD14 and osteocalcin, high levels of HLA, and no CD34, CD90 and nestin expression. FIG. 6 presents images of cell stained for the stem cell-specific markers, HES-1, SSEA4 and CD1 17, at day 5.
TABLE-US-00005 TABLE 5 Antibodies Used For Immunofluorescence Experiments Catalog Antigen Clone Isotype Vendor Number Concentration CD3 UCHT-1 Mouse IgG1 Pharmingen 555330 1:100 CD11b (MAC-1) M1/70 Rat IgG2b Chemicon MAB1387Z 1:100 CD14 UCHM-1 Mouse IgG2a Chemicon CBL453 1:100 CD34 581 Mouse IgG1k Pharmingen 555820 1:100 CD45 69 Mouse IgG1 BD Transduction Labs 610266 1:100 CD90 (Thy-1) F15-42-1 Mouse IgG1 Chemicon CBL415 1:100 CD117 (c-kit) YB5.B8 Mouse IgG1 Chemicon MAB1162 1:100 a-fetoprotein 2X2 Mouse IgG2a USBiological F4100-04 1:100 C-Peptide C-PEP-01 Mouse IgG1 Chemicon CBL94 1:100 Cytokeratin-7 OV-TL 12/30 Mouse IgG1 Chemicon MAB3554 1:100 filter each time E-cadherin 67A4 Mouse IgG1 Chemicon MAB3199 1:100 Glut-2 Polyclonal Rabbit Chemicon AB1342 1:500 HES-1 Polyclonal Rabbit Chemicon AB5702 1:200 HLAabc 22.64.4 aka PHM-4 Mouse IgG2b Chemicon MAB1275 1:100 Human Islet Cells 3D3 Mouse IgM Cymbus CBL400 1:100 MAP-2 Mouse IgG1 Chemicon MAB378 1:200 Nestin 10C2 Mouse IgG1 Chemicon MAB5326 1:200 NF Polyclonal Rabbit Chemicon AB1983 1:100 NSE 5E2 Mouse IgG2a Chemicon MAB324 1:100 Osteocalcin Polyclonal Rabbit Chemicon AB1857 1:200 Pankeratin AE1/AE3 Mouse IgG1 Chemicon MAB3412 1:100 Somatostatin Polyclonal Rabbit Chemicon AB5494 1:100 SSEA4 MC-813-70 Mouse IgG3 Chemicon MAB4304 1:100 VEGF-R3 Polyclonal Rabbit Chemicon AB1875 1:100 VEGF-R3 54703 Mouse IgG1 R&D Systems MAB3491 1:100 VEGFR (KDR) CH-11 Mouse IgG1 Chemicon MAB1667 1:100 vWF Polyclonal Rabbit Chemicon AB7356 1:50 vWF 21-43 Mouse IgG1 Chemicon MAB3442 1:100 Catalog Vendor Number Concentration Secondary Antibodies Donkey anti-MsIgG (H + L) F(ab)2 Cy-5 Jackson ImmunoResearch 715-176-150 1:100 Donkey anti-RbIgG (H + L) F(ab)2 FITC Jackson ImmunoResearch 711-096-152 1:100 Donkey anti-MsIgG (H + L) F(ab)2 FITC Jackson ImmunoResearch 715-096-150 1:100 Donkey anti Ms IgG Alexa488 Molecular Probes A21202 1:400 Goat anti Ms IgM Alexa488 Molecular Probes A21042 1:400 Goat anti-Rb IgG F(ab)2 Quantum Dot655 Chemicon AQ402-655 1:50 Goat anti-Ms IgG F(ab)2 Quantum Dot525 Chemicon AQ400-525 1:50 Goat anti-Rat IgG F(ab)2 Quantum Dot565 Chemicon AQ404-565 1:50 CounterStain DAPI Molecular Probes D21490 10 ug/ml
TABLE-US-00006 TABLE 6 Short-term phenotypic expression as revealed by immunofluorescence staining. Scope d2 d3 d4 d5 d6 d8 CD34 - - - - - - CD90 - - - - - - CD117 - - + + + ++ CD14 + + + + + + VEGF-R2 - - - - - ND VEGFR3 - - - - - ND Osteocalcin + + + ++ ++ +++ HLAabc +++ +++ +++ +++ +++ +++ CD11b + + + + + + CD45 ++ ++ ++ ++ ++ ++ HES-1 + + + ++ ++ ++ SSEA4 - - + ++ +++ +++
TABLE-US-00007 TABLE 7 Long-term phenotypic expression as revealed by immunofluorescence staining. Scope d10 d15 d19 d25 CD34 - - - - CD90 - - - - CD117 ++ ++ + + Nestin - - - - CD14 + + + + VEGF-R2 + + + + VEGFR3 ++ ++ ++ ++ Osteocalcin +++ +++ +++ +++ NF + + + + Glut-2 - - - - NSE + + + + MAP-2 - - - - HLAabc +++ +++ +++ +++ vWF - - - - Pankeratin - - - - CD11b + + + + CD45 ++ ++ ++ ++
Phenotypic Analysis: PCR
To further analyze the gene expression profile of these MDSCs, RT-PCR and quantitative PCR were performed. MDSCs were cultured from 1 to 25 days in de-differentiation medium and the expression of gene products from several different cell lineages were examined. For each time point, cells were collected (1×105 to 3×106 cells/well) and RNA was isolated using Qiagen Rneasy Kit (Cat. No. 74103) following the manufacturer's instructions. First strand cDNA was synthesized by mixing 1 ng-5 μg of RNA with 1 μl of 500 μg/ml of oligo(dT) (Invitrogen; catalog number 55063), 1 μl of 10 mM dNTPs (Invitrogen; catalog number 18427-013), and water to equal 12 μl. The mixture was heated to 65° C. for 5 minutes and the chilled on ice. Then 4 μl of 5× First-strand buffer, 1 μl of 0.1 M DTT (Invitrogen; catalog number 18427-013), 40 units of RNaseOUT (Invitrogen; catalog number 10777-019), and 200 units of Superscript III RNaseH.sup.- RT (Invitrogen; catalog number 18080-093) were added. The tube was gently mixed and incubated at 50° C. for 60 minutes. The tube was spun and the enzymes were inactivated by heating to 70° C. for 15 minutes. The concentration of cDNA was estimated using a spectrophotometer.
Primers were designed to amplify stem cell-specific, mesodermal, endothelial, neuronal, and pancreatic markers. Table 8 shows the primer sequences and sizes. Primers were designed by Primer3 software with TM=60° C. PCR reactions were performed in duplicate.
TABLE-US-00008 TABLE 8 PCR Primer Sequences Length SEQ ID Primer Name Sequence (5'-3') (bp) NO OCT4-F GAGAACAATGAGAACCTTCAGGAG 400 1 OCT4-R TTCTGGCGCCGGTTACAGAACCA 2 CD34-f ACCACTTCCCTCATCTCTCCTCCAA 434 3 CD34-R AGGGTGAGGGAGGCAGAGACAGAAA 4 KDR-F (VEGFR2) TGCAGGACCAAGGAGACTATGT 750 5 KDR-R (VEGFR2) TAGGATGATGACAAGAAGTAGCC 6 TIE-2-F ATCCCATTTGCAAAGCTTCTGGCTGGC 400 7 TIE-2-R TGTGAAGCGTCTCACAGGTCCAGGATG 8 CD31-F (PECAM1) AGGTCAGCAGCATCGTGGTCAACAT 800 9 CD31-R (PECAM1) GTGGGGTTGTCTTTGAATACCGCAG 10 VE-CADHERIN-F CTCTGCATCCTCACCATCACAG 250 11 VE-CADHERIN-R TAGCCGTAGATGTGCAGCGTGT 12 SM1-F TAAACACCTGCCCATCTACTCGG 350 13 SM1-R ATCTCATCATCCTGGGCTGCTGG 14 SM22A-F CGGCTGGTGGAGTGGATCATAG 400 15 SM22A-R CCCTCTGTTGCTGCCCATCTGA 16 PDGFRB-F GCCTTACCACATCCGCTC 200 17 PDGFRB-R TCACACTCTTCCGTCACATTGC 18 GATA4-F AGACATCGCACTGACTGAGAAC 200 19 GATA4-R GACGGGTCACTATCTGTGCAAC 20 NKX2.5-F CTTCAAGCCAGAGGCCTACG 840 21 NKX2.5-R CCGCCTCTGTCTTCTTCAGC 22 AFP-F TGCAGCCAAAGTGAAGAGGGAAGA 200 23 AFP-R CATAGCGAGCAGCCCAAAGAAGAA 24 ALB-F TGCTTGAATGTGCTGATGACAGGG 25 ALB-R AAGGCAAGTCAGCAGGCATCTCATC 26 CK18-F GTACTGGTCTCAGCAGATTGAGGAG 540 27 CK18-R GCTTCTGCTGGCTTAATGCCTCAGA 28 CK19-F ATGGCCGAGCAGAACCGGAA 330 29 CK19-R CCATGAGCCGCTGGTACTCC 30 GFAP-F TCATCGCTCAGGAGGTCCTT 31 GFAP-R CTGTTGCCAGAGATGGAGGTT 32 MAP2-F GAAGACTCGCATCCGAATGG 33 MAP2-R CGCAGGATAGGAGGAAGAGAC 34 MBP-F TTAGCTGAATTCGCGTGTGG 35 MBP-R GAGGAAGTGAATGAGCCGGTTA 36 GAD-F GCGCCATATCCAACAGTGACAG 37 GAD-R GCCAGCAGTTGCATTGACATATA 38 TAU-F GTAAAAGCAAAGACGGGACTGG 39 TAU-R ATGATGGATGTTGCCTAATGAG 40 TBX-5-F GCTGGAAGGCGGATGTTTC 41 TBX-5-R TCGTTTTGGGATTAATGCCC 42 SCF-F TGGTGGCATCTGACACTAGTGA 200 43 SCF-R CTTCCAGTATAAGGCTCCAAAAGC 44 BMP-4-F AGGAAGCAGTCTGTGTAGTGTG 170 45 BMP-4-R GATGGTAGTAGAGGGATGTGGG 46 SOX-2-F CTTGGGCAGGCTGATAGTTTTTA 47 SOX-2-R TTTGTACTTGGCTCATTGCTCCT 48 ABCG2-F TAGTTAATCTCCTCAGACAGTAA 161 49 ABCG2-R GCTACTAACCTACCTATTCATTT 50 NESTIN-F AGAGGGGAATTCCTGGAG 500 51 NESTIN-R CTGAGGACCAGGACTCTCTA 52 PDX-1-F AACGCCACACAGTGCCAAAT 142 53 PDX-1-R GCATGGGTCCTTGTAAAGCT 54 DPPA5 ATAAGCTTGATCTCGTCTTCC 220 55 DPPA5 CTTGCTAGGATGTAACAAAGC 56 ANF-F GACAGACTGCAAGAGGCTCC 57 ANF-R GGAGAGGCGAGGAAGTCACC 58 α-MYOSIN HEAVY AAGTTCCGCAAGGTGCAG 59 CHAIN-F α-MYOSIN HEAVY TTGGCAAGCAGTGAGGTTC 60 CHAIN-R MYOSIN LIGHT CCTTCCGCATGTTTGACC 61 CHAIN 2A-F MYOSIN LIGHT GCCCCTCATTCCTCTTTCTC 62 CHAIN 2A-R TROPONIN-F CAAAGATCTGCTCCTCGCTC 63 TROPONIN-R AGTGGTGGCTCCCACCTAG 64 ATP2A2-F AAGCCAATTTTTCTGCACTG 65 ATP2A2-R AACAATGTTTTCTGCACAAGC 66 BNP-F GCCTTTTGATACTCTTACTGTGGC 67 BNP-R CAGGAGAAAGATTGGGAAGTGG 68 C-KIT-F CCAAGTCATTGTTGGATAAG 200 69 C-KIT-R CTTAGATGAGTTTTCTTTCAC 70 CD13-F CCAGTCTAGTTCCTGATGACCC 71 CD13-R CAAGGCCGTTCATTGTCC 72 CD105-F AGTCAGCTCAGCAGCAG 73 CD105-R GGGGTCAACACCACAG 74 CD133-F ATCAGAACTGCAATCTGCACA 75 CD133-R AGAAGATCCCTGTCACAATTCC 76 REX-1-F CGCCTGTAGTCCCAGCTAC 188 77 REX-1-R GATCTTGGCTCACTGCAAGC 78 B-ACTIN-F GCACTCTTCCAGCCTTCCTTCC 79 B-ACTIN-R TCACCTTCACCGTTCCAGTTTTT 80 osteopontin-f CTAGGCATCACCTGTGCCATACC 600 81 osteopontin-r CAGTGACCAGTTCATCAGATTCATC 82 col2a1-f CCAGGACCAAAGGGACAGAAAG 83 col2a1-r TTCACCAGGTTCACCAGGATTG 84 PPAR2-f GCTGTTATGGGTGAAACTCTG 85 PPAR2-r ATAAGGTGGAGATGCAGGCTC 86 hIns-f GCCTTTGTGAACCAACACCTG 87 hIns-r GTTGCAGTAGTTCTCCAGCTG 88 IPF1-f CCCATGGATGAAGTCTACC 800 89 IPF1-r GTCCTCCTCCTTTTTCCAC 90 Ngn3-f CTCGAGGGTAGAAAGGATGACGCCTC 91 Ngn3-r ACGCGTGAATGGGATTATGGGGTGGTG 92 TAL-1-f ATGGTGCAGCTGAGTCCTCC 93 TAL-1-r TCTCATTCTTGCTGAGCTTC 94 GATA-2-f AGCCGGCACCTGTTGTGCAA 95 GATA-2-r TGACTTCTCCTGCATGCACT 96 Flk-1-f ATGCACGGCATCTGGGAATC 250 97 Flk-1-r GCTACTGTCCTGCAAGTTGCTGTC 98 GATA-3-f ACCCCACTGTGGCGGCGAGAT 99 GATA-3-r CACAGCACTAGAGACC 100 AC133-f CAGTCTGACCAGCGTGAAAA 400 101 AC133-r GGCCATCCAAATCTGTCCTA 102 INSULIN-F GCTGGTTCAAGGGCTTTATTC 218 103 INSULIN-R TGGGGCAGGTGGAGCTGGGCG 104 GAPDH-F AGGGGTCTACATGGCAACTG 228 105 GADPH-R CGACCACTTTGTCAAGCTCA 106 PAX-4-F TTSCCAGGCAAAGAGGGCTGGAC 153 107 PAX-4R GGCTGTGTGAGCAAGATCCTAGG 108 IAPP-F TAACAGTGCCCTTTTCATCTCC 217 109 IAPP-R(ISLET CTGTGCCACTGAGATATAGGTCC 110 AMYLOID POLYPEPTIDE GLUT2-F AAACAAAGCAAATGTTCAGTGG 176 111 GLUT2-R TGGGTCCCCAAAAGCTTAG 112 NEUROGENIN-F TCAGCAGGCAATAGATTGGG 200 113 NEUROGENIN-R AAAGGAAAGGCCGTCTAGGG 114 CARBOXYPEPTIDASE- GATCTACCTAGTTTAATAGACCC 148 115 F CARBOXYPEPTIDASE- TGTACTAGTTGAGAAAGCTGAT 116 R IGF2-F AGTGAGCAAAACTGCCGC 214 117 IGF2-R GAAGATGCTGCTGTGCTTCC 118
GLUCAGON-F CTTCACAACATCACCTGCTAGC 246 119 GLUCAGON-R ACAGGTTGGGGTACTTCATCC 120 ISLET-1-F TGAAATCCTGGGTCTCTTGG 330 121 ISLET-1-R GCAATGCAAGAGCAAACAAA 122 PANCREATIC GACTTTCCAGCAGTCCCATA 123 AMYLASE-F PANCREATIC GTTTACTTCCTGCAGGGAAC 124 AMYLASE-R GATA-4(N)-F CTACAGGGGCACTTAACCCA 157 125 GATA-4(N)-R AGAGCTGAATCGCTCAGAGC 126 HLA-A-F ACTCTGGAAGGTTCTCATGTG 193 127 HLA-A-R AGGTGTCTCCATCTCTGTCTC 128 KERATIN-F CTTTTCGCGCGCCCAGCATT 129 KERATIN-R GATCTTCCTGTCCCTCGAG 130 E-CADHERIN-F AGAACAGCACGTACACAGCC 131 ECADHERIN-R CCTCCGAAGAAACAGCAAGA 132 CD90(THY1)-F AGAAGGTGACCAGCCTAACGG 324 133 CD90(THY1)-R TCTGAGCACTGTGACGTTCTG 134 CD9-F GCTCTGGACAAACCCTGCA 250 135 CD9-R AGTGGGAGTCCAAGACTCAG 136 CD45-F ATTTATTTTGTCCTTCTCCCA 260 137 CD45-R GTTAACAACTTTTGTGTGCCAAC 138 GLP-1R-F TGAACCTGTTTGCATCCTTCA 139 GLP-1R-R ACTTGGCAAGCCTGCATTTGA 140 CD10-F TCAGTTTATCCTGCCCACTGATT 350 141 CD10-R GGGAGCTGATGAAACTCACAAAT 142 CD11B-F ACAGAGCTGCCTCTCGGTGGCCA 490 143 CD11B-R TTCCCTTCTGCCGGAGAGGCTACGC 144 CD33-F TAGCCCAGTCATTCCTAAACCAG 296 145 CD33-R CTGTCCTAAGAGGCAAGAAACCA 146 CD14-F AGGACTTGCACTTTCCAGCTTG 566 147 CD14-R TCCCGTCCAGTGTCAGGTTATC 148 CD38-F TTTTTAATGAGGTGGCTTTCTAACA 241 149 CD38-R AGCAATCCGAGGAAACGAG 150 CD4-F TCAGGGAAAGAAAGTGGTGC 138 151 CD4-R AAGAAGGAGCCCTGATTTCC 152 TROPONIN1-F TGATGTAGACGCTGCTGGTC 136 153 TROPONIN1-R GGCTCCAGCACCATGATACT 154 NSE-F CTGCTGATCCTTCCCGATAC 700 155 NSE-R ATTGGCTGTGAACTTGGACC 156 CXCR3-F CCACTGCCAATACAACTTCC 401 157 CXCR3-R GCAAGAGCAGCATCCACATC 158 CXCR4-F CATCTACACAGTCAACCTCTA 807 159 CXCR4-R CTAAAGAAACACAAGACAAAA 160 CDX-2F AGACCAACAACCCAAACAGC 151 161 CDX-2R GTCACCAGAGCTTCTCTGGG 162 HNF-3B-F AATCATTGCCATCGTGTG 262 163 HNF-3B-R CGCGGCTTAAAATCTGGTAT 164 NKX-2.2F TGGACGCTGTGCAGAGCCTG NA 165 NKX-2.2R CAGGTCCTGGGCTTTGAGCG 166 PAX6-F AACTGGAACTGACACACCAGG 191 167 PAX6-R CCTATGCAACCCCCAGTCC 168 OSTEOCALCIN-F CAGTTCTGCTCCTCTCCAGG 185 169 OSTEOCALCIN-R CCATCCTCCTGACACCTCC 170 GENESIS-F GCATCTGCGAGTTCATCAGCAAC 157 171 GENESIS-R GGGTCCAGGGTCCAGTAGTTGC 172 CD34-2F CCTGCTCTCTTGTAATGATATAGCC 227 173 CD34-2R GAGACTAGAACTGAGCTGTTTGTCC 174
For RT-PCR, 30-300 ng of cDNA was mixed with PHUSION HF buffer, PHUSION dNTPs, MgCl2, 200 nM of each primer, and PHUSION DNA polymerase (Finnzymes). The cycling parameters were 98° C. for 30 sec, followed by 40 cycles of 98° C. for 10 sec, 58-72° C. for 10 sec, 72° C. for 20 sec 2, and a final extension at 72° C. for 5 minutes. The products were resolved in 1-3% agarose gels.
For real time (quantitative) PCR, 100 ng of cDNA was mixed with 200 nM of each primer, and 0.5 volume of SYBR green qPCR SuperMix-UDG with ROX (Invitrogen; catalog number 11744). The cycling parameters were 50° C. for 2 minutes, 95° C. for minutes, followed by 40 cycles of 60° C. for 30 seconds and 95° C. for 30 seconds. To determine the relative gene expression, the ΔCT values for controls (GADPH and β-actin) were compared to pancreatic gene expression. To calculate the percent of relative expression the following formula was used:
R.E. (relative expression)=2n-(ΔCT gene-ΔCT GAPDH)×100
Tables 9-16 and FIG. 8 present the results of the PCR analyses. Expression of the stem cell-specific markers, OCT-4, CD117, DPPA5, SCF, and Genesis, was increased in the de-differentiated stem cells relative to the undifferentiated monocytes.
TABLE-US-00009 TABLE 9 Expression of Stem Cell Markers Days in Culture Marker 1 5 10 19 OCT-4 - - + + CD117 - + + + DPPA5 - + + + SCF - - + +
TABLE-US-00010 TABLE 10 Expression of Embryonal Carcinoma (EC) Markers Days in Culture Marker 1 5 10 19 Flk-1 - - + + TIE-2 - + + + CD31 + + + + VE-cadherin - - - +
TABLE-US-00011 TABLE 11 Expression of Hematopoietic Markers Days in Culture Marker 1 5 10 19 CD34 + + + + TAL-1 - - - + GATA-2 - - - + CD133 + + + + GATA-3 - - - + AC133 + + + +
TABLE-US-00012 TABLE 12 Expression of Cardiac Markers Days in Culture Marker 1 5 10 19 GATA4 + + + + NKX2.5 - + + + NKX2.2 - - + + ANF + + + + BNP + + + + CD105 - - + + TBX-5 - + + + BMP-4 + + + +
TABLE-US-00013 TABLE 13 Expression of Pancreatic Markers Days in Culture Marker 1 5 10 19 IPF1 - + + + PDX-1 + + + + Insulin nt - + + PAX-4 - - - + IAPP + + + + GLUT2 + + + + Neurogenin nt nt + + Carboxypeptidase nt nt nt + IGF2 - + + + Glucagon - - + + Islet-1 + + + +
TABLE-US-00014 TABLE 14 Expression of Endodermal Markers Days in Culture Marker 1 5 10 19 AFP - + + + ALB - - - + CK19 + + + + CK18 + + + +
TABLE-US-00015 TABLE 15 Expression of Smooth Muscle Markers Days in Culture Marker 1 5 10 19 SM1 - - - + SM22A - - - + PDGFRB - + - +
TABLE-US-00016 TABLE 16 Expression of Cell Surface Markers Days in Culture Marker 1 5 10 19 CD4 + + + + CD9 + + + +
174124DNAArtificial Sequenceprimer 1gagaacaatg agaaccttca ggag 24223DNAArtificial Sequenceprimer 2ttctggcgcc ggttacagaa cca 23325DNAArtificial Sequenceprimer 3accacttccc tcatctctcc tccaa 25425DNAArtificial Sequenceprimer 4agggtgaggg aggcagagac agaaa 25522DNAArtificial Sequenceprimer' 5tgcaggacca aggagactat gt 22623DNAArtificial Sequenceprimer 6taggatgatg acaagaagta gcc 23727DNAArtificial Sequenceprimer 7atcccatttg caaagcttct ggctggc 27827DNAArtificial Sequenceprimer 8tgtgaagcgt ctcacaggtc caggatg 27925DNAArtificial Sequenceprimer 9aggtcagcag catcgtggtc aacat 251025DNAArtificial Sequenceprimer 10gtggggttgt ctttgaatac cgcag 251122DNAArtificial Sequenceprimer 11ctctgcatcc tcaccatcac ag 221222DNAArtificial Sequenceprimer 12tagccgtaga tgtgcagcgt gt 221323DNAArtificial Sequenceprimer 13taaacacctg cccatctact cgg 231423DNAArtificial Sequenceprimer 14atctcatcat cctgggctgc tgg 231522DNAArtificial Sequenceprimer 15cggctggtgg agtggatcat ag 221622DNAArtificial Sequenceprimer 16ccctctgttg ctgcccatct ga 221718DNAArtificial Sequenceprimer 17gccttaccac atccgctc 181822DNAArtificial Sequenceprimer 18tcacactctt ccgtcacatt gc 221922DNAArtificial Sequenceprimer 19agacatcgca ctgactgaga ac 222022DNAArtificial Sequenceprimer 20gacgggtcac tatctgtgca ac 222120DNAArtificial Sequenceprimer 21cttcaagcca gaggcctacg 202220DNAArtificial Sequenceprimer 22ccgcctctgt cttcttcagc 202324DNAArtificial Sequenceprimer 23tgcagccaaa gtgaagaggg aaga 242424DNAArtificial Sequenceprimer 24catagcgagc agcccaaaga agaa 242524DNAArtificial Sequenceprimer 25tgcttgaatg tgctgatgac aggg 242625DNAArtificial Sequenceprimer 26aaggcaagtc agcaggcatc tcatc 252725DNAArtificial Sequenceprimer 27gtactggtct cagcagattg aggag 252825DNAArtificial Sequenceprimer 28gcttctgctg gcttaatgcc tcaga 252920DNAArtificial Sequenceprimer 29atggccgagc agaaccggaa 203020DNAArtificial Sequenceprimer 30ccatgagccg ctggtactcc 203120DNAArtificial Sequenceprimer 31tcatcgctca ggaggtcctt 203221DNAArtificial Sequenceprimer 32ctgttgccag agatggaggt t 213320DNAArtificial Sequenceprimer 33gaagactcgc atccgaatgg 203421DNAArtificial Sequenceprimer 34cgcaggatag gaggaagaga c 213520DNAArtificial Sequenceprimer 35ttagctgaat tcgcgtgtgg 203622DNAArtificial Sequenceprimer 36gaggaagtga atgagccggt ta 223722DNAArtificial Sequenceprimer 37gcgccatatc caacagtgac ag 223823DNAArtificial Sequenceprimer 38gccagcagtt gcattgacat ata 233922DNAArtificial Sequenceprimer 39gtaaaagcaa agacgggact gg 224022DNAArtificial Sequenceprimer 40atgatggatg ttgcctaatg ag 224119DNAArtificial Sequenceprimer 41gctggaaggc ggatgtttc 194220DNAArtificial Sequenceprimer 42tcgttttggg attaatgccc 204322DNAArtificial Sequenceprimer 43tggtggcatc tgacactagt ga 224424DNAArtificial Sequenceprimer 44cttccagtat aaggctccaa aagc 244522DNAArtificial Sequenceprimer 45aggaagcagt ctgtgtagtg tg 224622DNAArtificial Sequenceprimer 46gatggtagta gagggatgtg gg 224723DNAArtificial Sequenceprimer 47cttgggcagg ctgatagttt tta 234823DNAArtificial Sequenceprimer 48tttgtacttg gctcattgct cct 234923DNAArtificial Sequenceprimer 49tagttaatct cctcagacag taa 235023DNAArtificial Sequenceprimer 50gctactaacc tacctattca ttt 235118DNAArtificial Sequenceprimer 51agaggggaat tcctggag 185220DNAArtificial Sequenceprimer 52ctgaggacca ggactctcta 205320DNAArtificial Sequenceprimer 53aacgccacac agtgccaaat 205420DNAArtificial Sequenceprimer 54gcatgggtcc ttgtaaagct 205521DNAArtificial Sequenceprimer 55ataagcttga tctcgtcttc c 215621DNAArtificial Sequenceprimer 56cttgctagga tgtaacaaag c 215720DNAArtificial Sequenceprimer 57gacagactgc aagaggctcc 205820DNAArtificial Sequenceprimer 58ggagaggcga ggaagtcacc 205918DNAArtificial Sequenceprimer 59aagttccgca aggtgcag 186019DNAArtificial Sequenceprimer 60ttggcaagca gtgaggttc 196118DNAArtificial Sequenceprimer 61ccttccgcat gtttgacc 186220DNAArtificial Sequenceprimer 62gcccctcatt cctctttctc 206320DNAArtificial Sequenceprimer 63caaagatctg ctcctcgctc 206419DNAArtificial Sequenceprimer 64agtggtggct cccacctag 196520DNAArtificial Sequenceprimer 65aagccaattt ttctgcactg 206621DNAArtificial Sequenceprimer 66aacaatgttt tctgcacaag c 216724DNAArtificial Sequenceprimer 67gccttttgat actcttactg tggc 246822DNAArtificial Sequenceprimer 68caggagaaag attgggaagt gg 226920DNAArtificial Sequenceprimer 69ccaagtcatt gttggataag 207021DNAArtificial Sequenceprimer 70cttagatgag ttttctttca c 217122DNAArtificial Sequenceprimer 71ccagtctagt tcctgatgac cc 227218DNAArtificial Sequenceprimer 72caaggccgtt cattgtcc 187317DNAArtificial Sequenceprimer 73agtcagctca gcagcag 177416DNAArtificial Sequenceprimer 74ggggtcaaca ccacag 167521DNAArtificial Sequenceprimer 75atcagaactg caatctgcac a 217622DNAArtificial Sequenceprimer 76agaagatccc tgtcacaatt cc 227719DNAArtificial Sequenceprimer 77cgcctgtagt cccagctac 197820DNAArtificial Sequenceprimer 78gatcttggct cactgcaagc 207922DNAArtificial Sequenceprimer 79gcactcttcc agccttcctt cc 228023DNAArtificial Sequenceprimer 80tcaccttcac cgttccagtt ttt 238123DNAArtificial Sequenceprimer 81ctaggcatca cctgtgccat acc 238225DNAArtificial Sequenceprimer 82cagtgaccag ttcatcagat tcatc 258322DNAArtificial Sequenceprimer 83ccaggaccaa agggacagaa ag 228422DNAArtificial Sequenceprimer 84ttcaccaggt tcaccaggat tg 228521DNAArtificial Sequenceprimer 85gctgttatgg gtgaaactct g 218621DNAArtificial Sequenceprimer 86ataaggtgga gatgcaggct c 218721DNAArtificial Sequenceprimer 87gcctttgtga accaacacct g 218821DNAArtificial Sequenceprimer 88gttgcagtag ttctccagct g 218919DNAArtificial Sequenceprimer 89cccatggatg aagtctacc 199019DNAArtificial Sequenceprimer 90gtcctcctcc tttttccac 199126DNAArtificial Sequenceprimer 91ctcgagggta gaaaggatga cgcctc 269227DNAArtificial Sequenceprimer 92acgcgtgaat gggattatgg ggtggtg 279320DNAArtificial Sequenceprimer 93atggtgcagc tgagtcctcc 209420DNAArtificial Sequenceprimer 94tctcattctt gctgagcttc 209520DNAArtificial Sequenceprimer 95agccggcacc tgttgtgcaa 209620DNAArtificial Sequenceprimer 96tgacttctcc tgcatgcact 209720DNAArtificial Sequenceprimer 97atgcacggca tctgggaatc 209824DNAArtificial Sequenceprimer 98gctactgtcc tgcaagttgc tgtc 249921DNAArtificial Sequenceprimer 99accccactgt ggcggcgaga t 2110016DNAArtificial Sequenceprimer 100cacagcacta gagacc 1610120DNAArtificial Sequenceprimer 101cagtctgacc agcgtgaaaa 2010220DNAArtificial Sequenceprimer 102ggccatccaa atctgtccta 2010321DNAArtificial Sequenceprimer 103gctggttcaa gggctttatt c 2110421DNAArtificial Sequenceprimer 104tggggcaggt ggagctgggc g 2110520DNAArtificial Sequenceprimer 105aggggtctac atggcaactg 2010620DNAArtificial Sequenceprimer 106cgaccacttt gtcaagctca 2010723DNAArtificial Sequenceprimer 107ttsccaggca aagagggctg gac 2310823DNAArtificial Sequenceprimer 108ggctgtgtga gcaagatcct agg 2310922DNAArtificial Sequenceprimer 109taacagtgcc cttttcatct cc 2211023DNAArtificial Sequenceprimer 110ctgtgccact gagatatagg tcc 2311122DNAArtificial Sequenceprimer 111aaacaaagca aatgttcagt gg 2211219DNAArtificial Sequenceprimer 112tgggtcccca aaagcttag 1911320DNAArtificial Sequenceprimer 113tcagcaggca atagattggg 2011420DNAArtificial Sequenceprimer 114aaaggaaagg ccgtctaggg 2011523DNAArtificial Sequenceprimer 115gatctaccta gtttaataga ccc 2311622DNAArtificial Sequenceprimer 116tgtactagtt gagaaagctg at 2211718DNAArtificial Sequenceprimer 117agtgagcaaa actgccgc 1811820DNAArtificial Sequenceprimer 118gaagatgctg ctgtgcttcc 2011922DNAArtificial Sequenceprimer 119cttcacaaca tcacctgcta gc 2212021DNAArtificial Sequenceprimer 120acaggttggg gtacttcatc c 2112120DNAArtificial Sequenceprimer 121tgaaatcctg ggtctcttgg 2012220DNAArtificial Sequenceprimer 122gcaatgcaag agcaaacaaa 2012320DNAArtificial Sequenceprimer 123gactttccag cagtcccata 2012420DNAArtificial Sequenceprimer 124gtttacttcc tgcagggaac 2012520DNAArtificial Sequenceprimer 125ctacaggggc acttaaccca 2012620DNAArtificial Sequenceprimer 126agagctgaat cgctcagagc 2012721DNAArtificial Sequenceprimer 127actctggaag gttctcatgt g 2112821DNAArtificial Sequenceprimer 128aggtgtctcc atctctgtct c 2112920DNAArtificial Sequenceprimer 129cttttcgcgc gcccagcatt 2013019DNAArtificial Sequenceprimer 130gatcttcctg tccctcgag 1913120DNAArtificial Sequenceprimer 131agaacagcac gtacacagcc 2013220DNAArtificial Sequenceprimer 132cctccgaaga aacagcaaga 2013321DNAArtificial Sequenceprimer 133agaaggtgac cagcctaacg g 2113421DNAArtificial Sequenceprimer 134tctgagcact gtgacgttct g 2113519DNAArtificial Sequenceprimer 135gctctggaca aaccctgca 1913620DNAArtificial Sequenceprimer 136agtgggagtc caagactcag 2013721DNAArtificial Sequenceprimer 137atttattttg tccttctccc a 2113823DNAArtificial Sequenceprimer 138gttaacaact tttgtgtgcc aac 2313921DNAArtificial Sequenceprimer 139tgaacctgtt tgcatccttc a 2114021DNAArtificial Sequenceprimer 140acttggcaag cctgcatttg a 2114123DNAArtificial Sequenceprimer 141tcagtttatc ctgcccactg att 2314223DNAArtificial Sequenceprimer 142gggagctgat gaaactcaca aat 2314323DNAArtificial Sequenceprimer 143acagagctgc ctctcggtgg cca 2314425DNAArtificial Sequenceprimer 144ttcccttctg ccggagaggc tacgc 2514523DNAArtificial Sequenceprimer 145tagcccagtc attcctaaac cag 2314623DNAArtificial Sequenceprimer 146ctgtcctaag aggcaagaaa cca 2314722DNAArtificial Sequenceprimer 147aggacttgca ctttccagct tg 2214822DNAArtificial Sequenceprimer 148tcccgtccag tgtcaggtta tc 2214925DNAArtificial Sequenceprimer 149tttttaatga ggtggctttc taaca 2515019DNAArtificial Sequenceprimer 150agcaatccga ggaaacgag 1915120DNAArtificial Sequenceprimer 151tcagggaaag aaagtggtgc 2015220DNAArtificial Sequenceprimer 152aagaaggagc cctgatttcc 2015320DNAArtificial Sequenceprimer 153tgatgtagac gctgctggtc 2015420DNAArtificial Sequenceprimer 154ggctccagca ccatgatact 2015520DNAArtificial Sequenceprimer 155ctgctgatcc ttcccgatac 2015620DNAArtificial Sequenceprimer 156attggctgtg aacttggacc 2015720DNAArtificial Sequenceprimer 157ccactgccaa tacaacttcc 2015820DNAArtificial Sequenceprimer 158gcaagagcag catccacatc 2015921DNAArtificial Sequenceprimer 159catctacaca gtcaacctct a 2116021DNAArtificial Sequenceprimer 160ctaaagaaac acaagacaaa a 2116120DNAArtificial Sequenceprimer 161agaccaacaa cccaaacagc 2016220DNAArtificial Sequenceprimer 162gtcaccagag cttctctggg 2016318DNAArtificial Sequenceprimer 163aatcattgcc atcgtgtg 1816420DNAArtificial Sequenceprimer 164cgcggcttaa aatctggtat 2016520DNAArtificial Sequenceprimer 165tggacgctgt gcagagcctg 2016620DNAArtificial Sequenceprimer 166caggtcctgg gctttgagcg 2016721DNAArtificial Sequenceprimer 167aactggaact gacacaccag g 2116819DNAArtificial Sequenceprimer
168cctatgcaac ccccagtcc 1916920DNAArtificial Sequenceprimer 169cagttctgct cctctccagg 2017019DNAArtificial Sequenceprimer 170ccatcctcct gacacctcc 1917123DNAArtificial Sequenceprimer 171gcatctgcga gttcatcagc aac 2317222DNAArtificial Sequenceprimer 172gggtccaggg tccagtagtt gc 2217325DNAArtificial Sequenceprimer 173cctgctctct tgtaatgata tagcc 2517425DNAArtificial Sequenceprimer 174gagactagaa ctgagctgtt tgtcc 25
Patent applications by Brian S. Newsom, Spring, TX US
Patent applications by Donna R. Rill, The Woodlands, TX US
Patent applications by Glenn E. Winnier, The Woodlands, TX US
Patent applications by Jim C. Williams, The Woodlands, TX US
Patent applications in class Blood, lymphatic, or bone marrow origin or derivative
Patent applications in all subclasses Blood, lymphatic, or bone marrow origin or derivative